1. Field of the Invention
The present invention relates to a measurement method using an enzyme, and a reagent kit used for the measurement method.
2. Description of Related Art
There are many methods for measuring an analyte using an enzyme, and the methods are broadly divided into two types. In the first type, an enzyme having specificity for an analyte acts directly on the analyte, thereby detecting the analyte. Examples of the first type include the following: a method in which the analyte is glucose and the enzyme is glucose oxidase or glucose dehydrogenase; and a method in which the analyte is uric acid and the enzyme is uricase. In the second type, an enzyme acting on an analyte is used to produce a product derived from the analyte, and then an enzyme having specificity for the product acts on the product, thereby detecting the analyte. Examples of the second type include the following: a method in which the analyte is a glycated protein, the enzyme acting on the analyte is a protease, and the enzyme having specificity is a fructosyl amino acid oxidase (FAOD); a method in which the analyte is cholesterol, the enzyme acting on the analyte is cholesterol esterase, and the enzyme having specificity is cholesterol oxidase; and a method in which the analyte is creatinine, the enzyme acting on the analyte is creatininase, and the enzyme having specificity is sarcosine oxidase.
In the second type of method, the enzyme having specificity for the product and the enzyme acting on the analyte may be added in the following order: 1) the enzyme having specificity for the product is added after the enzyme acting on the analyte; 2) the enzyme having specificity for the product is added prior to the enzyme acting on the analyte; or 3) the enzyme acting on the analyte and the enzyme having specificity for the product are added simultaneously.
When the enzyme having specificity for the product is added after the enzyme acting on the analyte, since a product with which the enzyme having the specificity is to react has been produced in advance from the analyte, this method has the advantage of allowing the enzyme having specificity to react with the product quickly.
When the enzyme having specificity for a product is added prior to the enzyme acting on the analyte, this method has the advantage of removing a substance that impairs the measurement accuracy. If the product derived from the analyte that is to be produced by the enzyme acting on the analyte is already present as an endogenous substance in a sample, the endogenous substance can impair the measurement accuracy. The addition of the enzyme having specificity for a product prior to the enzyme acting on the analyte can remove the endogenous substance beforehand.
When the enzyme acting on the analyte and the enzyme having specificity for a product are added simultaneously, this method has the advantage of providing those enzymes as a single reagent.
A method for measuring a glycated protein using an enzyme has been known, in which a protease acts on the glycated protein to produce a glycated amino acid or a glycated peptide, and then a FAOD acts on the glycated amino acid or the glycated peptide, so that hydrogen peroxide is generated and measured.
Although several types of FAODs are used in accordance with their substrate specificities, all of them react only with a glycated amino acid or a short glycated peptide. For example, WO 2008-108385 and WO 2004-038034 disclose the FAOD that reacts with a glycated peptide comprising 6 amino acids. However, the FAOD that reacts with a glycated peptide having more than 6 amino acids and the FAOD that reacts directly with the glycated protein have not been known.
Therefore, in order to measure the glycated protein with the FAOD, the glycated protein needs be decomposed into a glycated amino acid or a glycated peptide having not more than 6 amino acids. The decomposition method includes, e.g., a chemical process using an acid or alkali. In view of the labor savings and convenience, the most effective decomposition method is to combine a protease and a glycated protein denaturing agent. In this method, e.g., a measurable degree of the glycated amino acid or the glycated peptide can be produced from the glycated protein in about 5 minutes.
The glycated protein is generated when a sugar such as glucose is bound to the N-terminal amino group or the side-chain amino groups of lysine and arginine in the middle of the protein. Therefore, the glycation occurs in a plurality of sites of the protein molecules. Accordingly, there are a great many types of glycated proteins in which any one or more than one of the N-terminal amino groups and/or the amino groups of lysine and arginine in the middle of the protein are glycated.
In the case of HbA1c in which only the N-terminus of the β-chain of hemoglobin is glycated, a method for specifically measuring only the glycated site (i.e., the glycated site at the N-terminus of the (β-chain) has been put to practical use. On the other hand, a method for measuring some of the glycated sites of the glycated protein has been generally employed. This is because measuring only one particular glycated site of lysine and arginine in the middle of the protein is not practical, since the protease should act specifically on the particular glycated site so that a glycated amino acid or a glycated peptide is produced only from that particular glycated site, and the rate of glycation of the individual sites may vary depending on the state of the protein.
It is known that a glycated amino acid is easily produced when a sugar such as glucose coexists with an amino acid. In order to increase the time during which the protease is acting on the glycated protein, the protease is preferably added first. On the other hand, however, a sample may contain a glycated amino acid. Therefore, it is desirable that such a glycated amino acid is eliminated from the sample by adding the FAOD prior to the protease so as to measure the amount of the glycated protein accurately. If one type of protease is added after the FAOD and is used to produce a glycated amino acid or a glycated peptide derived from all the glycated sites of the glycated protein, it is necessary that either the concentration of the protease is increased, a protease with a low specificity is used, or the protease acts for a long time.
Thus, the use of a plurality of types of proteases also has been considered. In the measurement of HbA1c, it is difficult for a protease to act directly on the N-terminus of the β-chain of hemoglobin. Therefore, a method using two types of proteases has been proposed. Examples of this method include the following: a method that uses an enzyme for liberating an amino acid having a glycated α-amino group and another protease (WO 2000-50579 and WO 2000-061732); a method that includes an enzymatic treatment with a serine carboxypeptidase and a suitable endoprotease or exoprotease (JP 2001-57897 A); a method that includes a treatment with a protease capable of cleaving the carboxyl group side of the third leucine from the N-terminus of the β-chain of hemoglobin, and subsequently a treatment with a protease capable of cutting histidyl leucine from the generated fructosyl valyl histidyl leucine (JP 2000-300294 A); and a method that combines Glu-C and another protease (JP 2005-110657 A).
In the measurement of glycated albumin or glycated hemoglobin using a plurality of types of proteases, e.g., the following methods have been disclosed: a method that causes a protease reaction and allows the other endoprotease or exoprotease to act before and after the protease reaction or at the same time as the protease reaction (JP 2004-333455 A); a method that uses an endoprotease, an exoprotease, etc. either individually or in combination (JP 1998-262695 A, JP 2005-172533 A, JP 2001-095598 A, WO 2005-049858, WO 2005-049857, and JP 2001-258593 A); and a method that uses a selectively decomposable protease and a general protease (WO 2002-006519).
It is easily expected that the protease also decomposes and inactivates the FAOD, which is a protein. The following methods have been disclosed to address the problem of the inactivation of the FAOD by the protease. WO 1998-048043 discloses a method that inactivates a protease before a FAOD treatment to prevent the inactivation of the FAOD by the protease. WO 2007-125779 discloses a method that uses a FAOD that is highly resistant to a protease. WO 2003-033729 discloses a method that makes good use of the inactivation of a FAOD by a protease.